Induced Low-density Lipoprotein Oxidation Research Articles

Free radical scavenging and anti-isolated human LDL oxidation activities of Butea superba Roxb. extract

BackgroundButea superba Roxb. (B. superba), is an herbal plant traditionally used for rejuvenation. Additionally, there have been reports on its antioxidant properties. Low-density lipoproteins (LDL) oxidation is the leading cause of cardiovascular diseases (CVDs). Natural products with antioxidant properties have the potential to inhibit LDL oxidation. However, no work has been done about the anti-isolated human LDL oxidation of B. superba extract (BSE). This study aimed to investigate the antioxidant potential of BSE and its ability to prevent isolated human (LDL) oxidation induced by free radical agents.MethodsThe antioxidant properties were investigated by antioxidant assays, including 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), ferric reducing ability power (FRAP), nitric oxide (NO) and peroxynitrite scavenging assay. More so, anti-isolated human LDL oxidation activities were evaluated by 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and 3-morpholinosydnonimine hydrochloride (SIN-1) induced LDL oxidation assay.ResultsBSE exhibited a significant (p < 0.05) antioxidant activity in all the test systems, demonstrating its potential as a potent free radical scavenger. It displayed scavenging effects on DPPH (p < 0.05; IC50 = 487.67 ± 21.94 µg/ml), ABTS (p < 0.05; IC50 = 30.83 ± 1.29 µg/ml). Furthermore, it generated significantly (p < 0.05) increased antioxidant capacity in a dose-dependent manner in FRAP assay and exhibited significantly (p < 0.01) higher percent NO scavenging activity than gallic acid. Besides, BSE at 62.5 µg/ml exhibited a considerable percent peroxynitrite scavenging of 71.40 ± 6.59% after a 2 h period. Moreover, BSE demonstrated anti-isolated human LDL oxidation activity induced by AAPH and SIN-1 (p < 0.05) and revealed scavenging activity similar to ascorbic acid (p > 0.05). Identifying the main constituents of BSE revealed the presence of genistein, daidzein, and biochanin A through Liquid Chromatography-Mass Spectrometer/Mass Spectrometer (LC–MS/MS) analysis.ConclusionThis is the first report that the presence of isoflavones in BSE could play an important role in its antioxidation and isolated human LDL oxidation scavenging properties. These findings suggest the potential for developing antioxidant herbal supplements. However, further studies must be investigated, including efficacious and safe human dosages.

Antioxidant potentials of furanodihydrobenzoxanthones from Artocarpus elasticus and their protection against oxLDL induced injury in SH-SY5Y cells

Exposure to reactive oxygen species (ROS) leads to the oxidation of low-density lipoproteins (LDL), converting them into oxidized ones (oxLDL), which are involved in the pathogenesis of Alzheimer's disease, suggesting a potential link between lipid dysregulation and neurodegenerative processes. Phenolic metabolites derived from Artocarpus elasticus root bark were found to possess significant antioxidant properties at three different radical scavenging assays, including 2,2-diphenyl-1-picrylhydrazyl (DPPH), oxygen radical absorbance capacity (ORAC), and thiobarbituric acid reactive substances (TBARS). Among them, furanodihydrobenzoxanthones (1-3) demonstrated notable protection against Cu2+ induced LDL oxidation, with IC50 values ranging from 0.9 to 2.9 μM in measurement of the malondialdehyde (MDA) production at TBARS and prolonged lag times (>180 min) in the generation of conjugated diene (CD). At a concentration of 10 μM, all three compounds (1-3) effectively protected against LDL oxidation as determined by relative electrophoretic mobility (REM). The most potent compound 1 defended human neuroblastoma SH-SY5Y cells from oxLDL-mediated dysfunction, including oxLDL-induced cytotoxicity, inhibited reactive oxygen species (ROS) formation, and enhancing mitochondrial membrane potential (ΔΨm). Individual components annotation in the ethylacetate extract was performed using LC-ESI-QTOF/MS, which serves as a chemotaxonomic marker for A. elasticus root barks.

The Bioactive Compound Contents and Potential Protective Effects of Royal Jelly Protein Hydrolysates against DNA Oxidative Damage and LDL Oxidation.

In this study, the inhibition of DNA oxidative damage and low-density lipoprotein (LDL) oxidation of royal jelly protein (RJP) hydrolysates obtained from two commercial proteases were investigated. The results showed that the inhibition of DNA oxidative damage induced by the Fenton reaction, RJP, RJPs hydrolyzed by alcalase (RJP-A), RJPs hydrolyzed by flavourzyme (RPJ-F) and RJP two-stage hydrolysates (RPJ-AF) all had the effect of inhibiting deoxyribose oxidative damage. The inhibition effect of RJP, RJP-A, RJP-F and RJP-AF (1.0 mg/mL) were 47.06%, 33.70%, 24.19% and 43.09%, respectively. In addition, studies have also found that both RJP and RJP hydrolysates can reduce the production of 8-OH-2′-dG and the order of its inhibitory ability is RJP-AF ≒ RJP-A > RJP-F > RJP. The inhibition of DNA damage induced by bleomycin-Fe3+/ascorbic acid (Asc) with the addition of RJP, RJP-A, RPJ-F and RPJ-AF were 17.16%, 30.88%, 25.00% and 37.25%, respectively. The results of LDL oxidation inhibition showed that RJP-AF (1 mg/mL) not only had the most effective inhibitory Cu2+-induced LDL oxidation to produce a thiobarbituric acid reactive substance (TBARS) but also extended the lag time of conjugated diene formation to 300 min, which was 3.3 times that of the control group.

Antioxidative and Inhibitory Effects of the Fruiting Body of Black Lingzhi Mushroom, Amauroderma rugosum (Agaricomycetes), on LDL Oxidation and HMG-CoA Reductase Activity.

Amauroderma rugosum fruiting bodies possess excellent cardiovascular benefits, including antioxidative, antihyperlipidemic, antihypertensive, antiinflammatory, anti-platelet aggregation, and antithrombotic effects. In this article, we describe our investigations of the in vitro antioxidant activity and in vitro antiatherosclerotic potential through inhibitory effects on low-density lipoprotein (LDL), LDL peroxidation, and 3-hydroxy3-methylglutaryl-coenzyme A (HMG-CoA) reductase catalytic activity using various fruiting body extracts partitioned with an organic solvent. Among 5 extracts/fractions tested, the semipolar ethyl acetate (EA) fraction demonstrated good antioxidant capacity based on total phenolic content, 2,2-diphenyl-1-picrylhydrazyl free radical scavenging, ferrous ion-chelating ability, cupric ion-reducing antioxidant capacity, and lipid peroxidation assays. The EA fraction also showed the strongest inhibitory effect on Cu2+-induced LDL oxidation via thiobarbituric acid reactive substances formation and HMG-CoA reductase activity. Chemical analysis conjointly identified 10 phenolic compounds (4 benzoic acid derivatives, 3 flavonoids, 1 cinnamic acid, 1 hexahydroxydiphenic acid dilactone, and 1 xanthone derivative), some of which play pivotal roles in arresting the physiopathogenesis of atherosclerosis, thereby attenuating the risk of cardiovascular events occurring.

Antioxidative properties and ability of phenolic compounds of Myrtus communis leaves to counteract in vitro LDL and phospholipid aqueous dispersion oxidation.

Antioxidant activities of Myrtus communis leaf phenolic compounds (McPCs) were investigated on 2,2'-9-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS(+) •) and oxygen radical absorbance capacity (ORAC) tests or on oxidation of biological models, human low-density lipoprotein (LDL) and phospholipid aqueous dispersion (L-α-phosphatidylcholine stabilized by bile salts). Two extraction techniques, microwave-assisted (MAE) and conventional (CE), were used to isolate McPCs, producing similar results of phenolic compound content. ABTS(+) • assay showed clearly that myrtle extracts exhibited a stronger scavenging effect than butylated hydroxyanisole and α-tocopherol, with a slight advantage for myrtle CE extract. In ORAC assay, the both McPC extracts were similarly less effective than the pure compounds as caffeic acid and myricitrin (myricetin 3-O-rhamnoside) but stronger than butylated hydroxytoluene. Moreover, myrtle CE and MAE extracts, and myricitrin were able to inhibit similarly the production of conjugated dienes and to prolong the lag phase (Tlag) during Cu(2+)-induced LDL oxidation with a dose-response effect. The cryo-electron microscopy observations on studied phospholipid dispersion stabilized by bile salts (BS) revealed the presence of bilayer vesicles and micelles. In 2,2'-azobis (2-amidinopropane) hydrochloride-induced phospholipid/BS oxidation, myrtle CE and MAE extracts gave similar effects to α-tocopherol and caffeic acid but myricitrin showed a higher protective effect than myrtle extracts. We showed also that no synergic or additive effect between α-tocopherol and myrtle extracts or caffeic acid in α-tocopherol-enriched phospholipid/BS dispersion, but myricitrin showed an additive effect and thus promoted the total antioxidant activity. These data showed that myrtle extract could be used as potential natural antioxidants, food stabilizers, or natural health products. We show that microwave-assisted extraction could be an alternative method for plant phenolic compound recovery allowing important gain in time extraction.We report inhibition of low-density lipoprotein oxidation in vitro initiated by Cu(2+) ions. We report that myrtle extract may be a source of natural antioxidants to counteract phospholipid peroxidation as well as α-tocopherol.

Simultaneous quantification and inhibitory effect on LDL oxidation of the traditional Korean medicine, Leejung-tang

BackgroundLeejung-tang (LJT) is a traditional Korean herbal medicine for the treatment of gastrointestinal disorders. In this study, we performed quantification analysis of five marker components, liquiritin (1), ginsenoside Rg1 (2), ginsenoside Rb1 (3), glycyrrhizin (4), and 6-gingerol (5) in LJT using a high performance liquid chromatography-photodiode array (HPLC–PDA). In addition, we investigated the inhibitory effect on low-density lipoprotein (LDL) oxidation by the LJT sample.MethodsCompounds 1–5 were separated within 35 min using a Gemini C18 column. The mobile phase used gradient elution with 1.0% (v/v) aqueous acetic acid (A) and 1.0% (v/v) acetic acid in acetonitrile (B). The flow rate was 1.0 mL/min and the detector was a photodiode array (PDA) set at 203 nm, 254 nm, and 280 nm. The inhibitory effect on LDL oxidation conduct an experiment on thiobarbituric acid reactive substance (TBARS) assay, relative electrophoretic mobility (REM) assay, and electrophoresis of ApoB fragmentation of LJT.ResultsCalibration curves of compounds 1–5 showed good linearity (r2 ≥0.9995) in different concentration ranges. The recoveries of compounds 1–5 were in the range of 98.90–103.39%, with relative standard deviations (RSD) below 3.0%. The RSDs (%) of intra-day and inter-day precision were 0.10–1.08% and 0.29–1.87%, respectively. The inhibitory effect of LJT on Cu2+-induced LDL oxidation was defined by TBARS assay (IC50: 165.7 μg/mL) and REM of oxLDL (decrease of 50% at 127.7 μg/mL). Furthermore LJT reduced the fragmentation of ApoB of oxLDL in a dose-dependent manner.ConclusionsThe established HPLC-PDA method will be helpful to improve quality control of LJT. In addition, LJT is a potential LDL oxidation inhibitor.

Disubstituted diaryl diselenides as potential atheroprotective compounds: Involvement of TrxR and GPx-like systems

Oxidative modifications of low-density lipoproteins (LDLs) have a determinant role in atherogenesis and the study of agents that can modulate LDL oxidation is of pharmacological and therapeutic significance. Therefore, the aim of this study was to evaluate the antioxidant effect of the disubstituted diaryl diselenides, p-methoxyl-diphenyl diselenide (p-CH3O–C6H4Se)2 (DM) and p-chloro-diphenyl diselenide (p-Cl–C6H4Se)2 (DC), on Cu2+-induced LDL oxidation. Both compounds caused a dose-dependent inhibition of human serum and isolated LDL oxidation evidenced by the increasing of the lag phase of lipid peroxidation and decreased the lipid oxidation rate (Vmax). The protein moieties from isolated LDL were also protected from Cu2+-induced oxidation. Moreover, the disubstituted diaryl diselenides efficiently decreased the oxidized LDL (ox-LDL) induced foam cell formation in J774A.1 macrophage cells. Mechanistically, we have demonstrated that the antioxidant and antiatherogenic effects of DM and DC are related to formation of their selenol intermediates (RSeH) either by a direct reaction with endogenous thiols (GPx-like activity) or via their reduction by TrxR (using NADPH as electron donor). Considering the powerful effect of DM and DC against LDL-induced toxicity, they could be considered for developing of new therapeutic approaches to preventing and treating atherosclerosis and cardiovascular diseases.

Inhibitory activity of Ecklonia stolonifera and its isolated phlorotannins against Cu2+-induced low-density lipoprotein oxidation

Since the oxidation of low-density lipoprotein (LDL) is one of the main causes of atherosclerosis, Cu2+-induced LDL oxidation and conjugated diene formation is currently being explored for the development of pharmaceutical drugs or functional foods for the treatment of atherosclerosis. The present work investigated the inhibitory effects on in vitro Cu2+-induced human LDL oxidation and conjugated diene formation of the methanol (MeOH) extract of the edible brown alga (Ecklonia stolonifera) and its different solvent-soluble fractions, as well as the phlorotannins isolated from them. The most active ethyl acetate fraction was selected for chromatographic separation to isolate six phlorotannins: phloroglucinol (1), dioxinodehydroeckol (2), eckol (3), phlorofucofuroeckol A (4), dieckol (5), and 7-phloroeckol (6). Compounds 3–6 showed potent inhibitory activity against Cu2+-induced LDL oxidation as compared with probucol, a well-known clinical therapeutic agent for hypercholesterolemia. Moreover, when compound 5 (at levels of 9 and 4.5 μM) was used in combination with probucol (4.5 μM), they additively inhibited Cu2+-induced LDL oxidation. In addition, 3–5 significantly prolonged the lag time of conjugated diene formation at 10 μM. These results suggest that the potent antiatherosclerotic effects of E. stolonifera and its isolated phlorotannins may be partly attributed to the inhibition of Cu2+-induced LDL oxidation and conjugated diene formation.

Thiosemicarbazone derivate protects from AAPH and Cu2+-induced LDL oxidation

AimsSeveral lines of evidence support the hypotheses that the oxidation of low density lipoprotein (LDL) may play a crucial role in the initiation and progression of atherosclerosis. Oxidative stress is one of the causes of the overproduction of reactive species that increase the formation of oxidized LDL. Thiosemicarbazones are compounds used in anticancer, antiviral and antifungal therapy; however, its redox activity has been controversial. Thus, we tested, in vitro, a possible antioxidant activity of a thiosemicarbazone derivate, the isatin-3-N4-benzilthiosemicarbazone (IBTC). Main methodsWe measured the conjugated diene formation in serum and LDL as well as the loss of tryptophan fluorescence in LDL induced by two oxidant agents, 2,2-azobis(2-amidinopropane dihydrochloride) (AAPH) and Cu2+. Thiobarbituric acid reactive substances (TBARS) formation in LDL and in different rat tissues was also assessed. The toxicity of IBTC was measured using aortic slices viability assay. Key findingsOur results show that IBTC significantly reduced the AAPH and Cu2+-induced formation of conjugated dienes, increased in a dose-dependent manner the lag phase and the t1/2 of tryptophan fluorescence, and reduced the TBARS formation in LDL, plasma and rat tissues, showing no toxicity to aortic slices. SignificanceThese results indicate that IBTC is a good antioxidant and a promising antiatherogenic agent for further studies in vivo.

The Inhibitory Effects of Fucoidans from &lt;i&gt;Laminaria japonica&lt;/i&gt; on Oxidation of Human Low-Density Lipoproteins

Five Fucoidan fractions from Laminaria japonica with different sulfate content and molecular weight were obtained by anion-exchange chromatography and mild acid hydrolysis. Their antioxidant effects on azo radicals AAPH- and AMVN -induced oxidation of human low-density lipoprotein (LDL) were evaluated by monitoring cholesteryl ester hydroperoxides (CHE-OOH) formation kinetics by HPLC analysis. Two antioxidants probucol and Vc were used as comparison. Fucoidan F-C with low molecular weight 2,000-8,000 and sulfate content 24.3% had much stronger protective antioxidant effects than other four Fucoidan fractions on both hydrophilic radical AAPH and lipophilic radical AMVN induced LDL oxidation. In AMVN induced LDL oxidation system, the other four Fucoidan fractions with molecular weight of 200,000 and 20,000 were able to modify the kinetics of LDL oxidation with the similar efficiency. However, the highly sulfated Fucoidan fraction L-B with molecular weight 20,000 was completely ineffective in protecting LDL from AAPH induced oxidation, whilst it effectively suppressed the oxidation of LDL induced by AMVN. The different protective antioxidant effects of Fucoidans on AAPH and AMVN induced LDL oxidation were due to their various structures and properties, and their capacities to interact with the different sites Fucoidans reacted on LDL.

Hemopexin Is Protective Against Intravascular Hemolysis–Induced Vascular Damage

AbstractAbstract 3207 Background:Intravascular hemolysis renders hemoglobin (Hb), usually protected by the red blood cell environment, susceptible to oxidation to its ferric (metHb) form. Haptoglobin (Hp) provides first aid to protect the vasculature from acellular metHb havoc. However, once Hp is consumed, ferrous Hb readily undergoes oxidation to metHb, in which the globin to heme affinity is reduced. MetHb releases the loosely bound heme allowing it to execute its oxidative activity. While nucleated cells, like endothelial cells, are equipped with the heme oxygenase-1 defence system, the nucleus lacking red blood cells (RBC) and low density lipoproteins (LDL) particles have no defence from heme-induced oxidation. Therefore the sole defence from heme oxidative damage remains the plasma protein hemopexin (Hx), which binds hemin with high affinity comparable to that of globin. Following hemin binding, Hx undergoes conformational changes rendering the heme inaccessible thereby inactive. Aim:The current study is focused on the ability of plasma Hx to defend the vasculature from acellular Hb induced damage. Results:Hx defence from metHb damage was studied in the following projects: a) preventing in vitro LDL oxidation by metHb, b) decreasing in vitro lysis of red cells created by hypotonic and mechanical stress. (c) Exploring consumption of Hx in hemodialysis (HD) treated patients by measuring their Hx levels. (a) Methemoglobin iron is a central cause of LDL oxidation. This process initiates formation of blood vessel plaques and atherosclerosis development. This part of the study examined the effect of apoHx (lacking heme) on LDL oxidation by metHb. Incubation of isolated native LDL (nLDL) or vitamin E depleted (dLDL) with low concentration of metHb range (1-10μM) resulted in covalent apoB inter-particle crosslinking in both LDL types, dLDL being more sensitive than nLDL to the oxidative crosslinking. Addition of apoHx abolished metHb-induced oxidation even in the dLDL. This Hx activity (comparable to that of haptoglobin) was confirmed by both SDS-PAGE and fluorescent assay of bityrosines formation. The fact that Hx protects LDL, especially vitamin E depleted particles, shed light on the controversial antioxidant vitamin E properties by indicating its efficacy in defence against acellular Hb induced LDL oxidation. (b) Like LDL the red cell membrane is exposed to metHb-induced oxidative damage. Acellular Hb was formed by hypotonic shock or mechanical shear stress to fresh whole blood of healthy donors. The effect of Hx on acellular Hb levels was measured spectrophotometrically. Reduction of osmotic pressure in fresh blood to 40% of its normal value resulted in formation of ∼300 μM acellular Hb. Supplementation of exogenous apoHx (20μM) to blood before exposure to hypotonicity reduced acellular Hb formation by half. The specificity of Hx was proved by addition of up to 80μM albumin, which had no effect on acellular Hb concentration. Acellular Hb was formed also by applying shear stress on circulating fresh blood in narrow channels using a peristaltic pump. Within 4 hours acellular Hb reached ∼500μM. However, in presence of 20μM apoHx hemolysis was significantly reduced (up to 50%). This reduction may be attributed to Hx's ability to block the hemolysis amplifying effect of metHb on the RBC membrane. (c) In HD treated patients RBCs are exposed to the effects of recurrent mechanical shear stress. The last part of the research was dedicated to quantisation of apoHx levels (by titration with hemin) in sera of HD patients (n=60) prior to and immediately after a single dialysis procedure versus normal controls (n=20). Sera apoHx levels in HD patients prior to a dialysis session were lower compared to healthy controls (10.91±3.19 μM vs. 14.95±2.63 μM, p<0.05) and were even lower (7.49±3.8 μM) immediately after the treatment. These findings indicate that in the plasma of HD treated patients Hx consumption rate exceeds synthesis rate leading to a chronically low level of Hx. Moreover, reduction of Hx was even more prominent in patients on long term HD treatment of above five years. Conclusion:Hx is specifically effective in attenuation of acellular Hb damage to the vasculature. These results point to the option that treating HD patients with exogenous Hx may slow down the rate of atherosclerotic cardiovascular disease development, the major cause of morbidity and mortality in these patients. Disclosures:No relevant conflicts of interest to declare.

Salvianolic acid B inhibits low-density lipoprotein oxidation and neointimal hyperplasia in endothelium-denuded hypercholesterolaemic rabbits

Atherosclerosis and restenosis are inflammatory responses involving free radicals and lipid peroxidation and may be prevented/cured by antioxidant-mediated lipid peroxidation inhibition. Salvianolic acid (Sal B), a water-soluble antioxidant obtained from a Chinese medicinal herb, is believed to have multiple preventive and therapeutic effects against human vascular diseases. In this study the in vitro and in vivo inhibitory effects of Sal B on oxidative stress were determined. In human aortic endothelial cells (HAECs), Sal B reduced oxidative stress, inhibited low-density lipoprotein (LDL) oxidation and reduced oxidised LDL-induced cytotoxicity. Sal B inhibited Cu(2+) -induced LDL oxidation in vitro (with a potency 16.3 times that of probucol) and attenuated HAEC-mediated LDL oxidation as well as reactive oxygen species (ROS) production. In cholesterol-fed New Zealand White rabbits (with probucol as positive control), Sal B intake reduced Cu(2+) -induced LDL oxidation, lipid deposition in the thoracic aorta, intimal thickness of the aortic arch and thoracic aorta and neointimal formation in the abdominal aorta. The data obtained in this study suggest that Sal B protects HAECs from oxidative injury-mediated cell death via inhibition of ROS production. The antioxidant activity of Sal B may help explain its efficacy in the treatment of vascular diseases.

Fenugreek Extract Rich in 4-Hydroxyisoleucine and Trigonelline Activates PPARα and Inhibits LDL Oxidation: Key Mechanisms in Controlling the Metabolic Syndrome

Fenugreek ( Trigonella foenum-graecum L.) has properties which are hypolipidemic, hypocholesterolemic, hypoglycemic, insulinotropic, and antioxidant. These properties are related to the control of the Metabolic Syndrome, a disorder of carbohydrate and lipid metabolism which increases the risk of diabetes and cardiovascular disease. The purpose of this study was to evaluate the effect of fenugreek extract (Hydroxylean®, Naturex) containing 25% of 4-hydroxyisoleucine (4-OH-Ile, isomer 2 S,3 R,4 S) and 15.7% of trigonelline on peroxisome proliferator-activated receptor alpha (PPARα) transcriptional activity and Cu2+-induced low-density lipoprotein (LDL) oxidation in vitro. The activation of PPARα is linked to controlling lipidemia, cholesterolemia and glycemia, all associated with diabetes and cardiovascular disease. Also, the inhibition of LDL oxidation reduces the risk of stroke. 1,000 μg/mL of fenugreek extract showed a 31.2% activation of PPARα as compared with the positive control, 100 μM of fenofibrate. Fenugreek extract also inhibited LDL oxidation induced by Cu2+ at concentrations higher than 5 μg/mL (P&lt;0.01). To our knowledge, this is the first report on PPARα transcriptional activity and Cu2+-induced LDL oxidation in vitro for fenugreek extract rich in 4-OH-Ile and trigonelline. The findings support the current evidence on fenugreek's capacity to control the Metabolic Syndrome.

In search of antioxidants and anti‐atherosclerotic agents from herbal medicines

Many recent studies have suggested that low-density lipoprotein (LDL) oxidation, endothelial dysfunction, and inflammation are involved in the pathogenesis of atherosclerosis. Herbal regimens in the treatment of blood stasis, a counterpart of atherosclerosis, commonly use medicinal plants of leguminosae and labiatae. We have developed disease-oriented screening methods to search for bioactive components, particularly isoflavones in leguminosae and polyphenols in labiatae from Chinese herbal medicines. Many bioactive components and active fractions capable of inhibiting a. Cu(II)-induced LDL oxidation, b. oxidized LDL-induced endothelial damage, c. uptake of oxidized LDL by macrophages (J774A.1), and d. expression of cell adhesion molecules (CAMs) have been identified. A polyphenol, namely salvianolic acid B from Salvia miltiorrhiza was identified to be a potent antioxidant, endothelial-protecting agent, and an inhibitor to suppress the expression of ICAM and VCAM. This review also briefly describes the strategy for developing herbal medicines as anti-atherosclerotic agents.

Changes in the Antioxidative Property of Herring (Clupea harengus) Press Juice during a Simulated Gastrointestinal Digestion

The aqueous fraction (press juice, PJ) from herring muscle was recently shown to inhibit hemoglobin-mediated oxidation of washed fish mince lipids during ice storage. As a first step to evaluate potential in vivo antioxidative effects from herring PJ, the aim of this study was to investigate whether herring PJ retains its antioxidative capacity during a simulated gastrointestinal (GI) digestion. Press juice from whole muscle (WMPJ) and light muscle (LMPJ) was mixed with pepsin solution followed by stepwise pH adjustments and additions of pancreatin and bile solutions. Digestive enzymes were removed from samples by ultrafiltration (10 kDa). Before, during, and after digestion, samples were analyzed for their peptide content and for antioxidative properties with the oxygen radical absorbance capacity (ORAC) and the low-density lipoprotein (LDL) oxidation assays. From 0 to 165 min of digestion, the content of <10 kDa peptides in WMPJ and LMPJ samples increased 12- and 7-fold, respectively. Further, both samples got approximately 12.5 times higher ORAC values and gave rise to approximately 1.3-fold increased lag phase in Cu2+-induced LDL oxidation. The largest changes in peptide content, ORAC values, and LDL oxidation inhibition occurred between 30 and 75 min of digestion, indicating that these parameters might be interrelated. When comparing analytical data obtained after 165 min of digestion with data obtained from analyses of native nondigested PJs, it was found that the data on peptide content, ORAC, and LDL oxidation from digested PJs were 64-69%, 121-161%, and 112-115%, respectively, of those of nondigested PJs. The study thus showed that enzymatic breakdown of PJ proteins under GI-like conditions increases the peroxyl radical scavenging activity and the potential to inhibit LDL oxidation of herring PJs. These data provide a solid basis for further studies of uptake and in vivo activities of herring-derived aqueous antioxidants.

Effect of Fast-Food Mediterranean-Type Diet on Human Plasma Oxidation

Oxidation of lipoproteins, particularly of low-density lipoprotein (LDL), is of prime importance in the initiation and progression of atherosclerosis. The Mediterranean diet has been associated with an unexpectedly low rate of cardiovascular events. Type 2 diabetic patients are at high risk of developing atherosclerosis. Functional alterations in the endothelium, which lead to atherosclerosis, are stimulated by oxidized lipoproteins, particularly oxidized LDL. The present study investigated the effect of Greek quick casual Mediterranean-type diet (fast food Mediterranean-type diet) consumption on the resistance to oxidation in plasma from type 2 diabetic patients and healthy human subjects. Lipids from fast food Mediterranean-type foodstuffs were extracted and tested in vitro for their ability to inhibit copper (Cu2+)-induced LDL oxidation. Foodstuffs that exerted the most potent in vitro antioxidative activity were chosen for the diet of study groups. Eighteen type 2 diabetic patients (group A) and 10 healthy subjects (group B) were fed a 4-week diet contained the chosen foodstuffs, while 17 type 2 diabetic patients (group C) were kept on their regular diet that they were following before the study. Type 2 diabetic patients were treated with sulfonylureas or metformin and were under good glycemic control (hemoglobin A1C < 7%). Serum lipoproteins, triglycerides, glucose, body mass index (BMI), and plasma resistance to Cu2+-induced oxidation before and after the 4-week diet were monitored. At the beginning of the study, no statistical difference was detected in plasma resistance to Cu2+-induced oxidation between type 2 diabetic patients (groups A and C) and healthy human subjects (group B), as this was detected at a time before the oxidation products become detectable, namely, lag time. After the 4-week period on the chosen diet the lag time in groups A and B significantly increased, while it was not changed in group C. In type 2 diabetic patients lag time was increased from 57.3 +/- 13.3 minutes (mean +/- SD) to 103.8 +/- 21.8 minutes (mean +/- SD) (P < .000), while in healthy human subjects there was an increase from 58.0 +/- 8.5 minutes (mean +/- SD) to 85.7 +/- 21.8 minutes (mean +/- SD) (P < .004). In all groups, total cholesterol, high-density lipoprotein-cholesterol, LDL-cholesterol, triglycerides, glucose, and BMI were not changed. Fast food Mediterranean foodstuffs exerted antioxidant activities both in vitro and in vivo after consumption in type 2 diabetic patients and healthy human subjects. Therefore consumption of a fast food Mediterranean-type diet should contribute to prevention against cardiovascular diseases.

The protective effect of Opuntia dillenii Haw fruit against low-density lipoprotein peroxidation and its active compounds

The antioxidant activity and inhibitory effect of extracts from Opuntia dillenii Haw fruit (ODHF) and its active compounds on low-density lipoprotein (LDL) peroxidation were investigated. The results indicated that the antioxidant activity of methanolic extracts of ODHF in Trolox equivalent antioxidant capacity and oxygen-radical absorbance capacity assays were in the order of seed > peel > pulp. The lag time of conjugated diene formation in Cu 2+-induced LDL oxidation was increased by incubation of LDL with various methanolic extracts of ODHF. The methanolic extracts from seed, peel and pulp prolonged the lag time compared to control (154.1 min) to 514.8, 163.9 and 190.2 min, respectively, at a concentration of 10 μg/ml. Among the extracts, seed extracts of ODHF (10 μg/ml) possessed the highest inhibitory effect on the formation of thiobarbituric acid reactive substances and relative electrophoretic mobility. The results also demonstrated that seeds of ODHF contained the highest amounts of polyphenols and flavonoids (212.8 and 144.1 mg/100 g fresh seed, respectively), such as gallic acid, catechin, sinapinic acid, epicatechin, p-coumaric acid, quercetin and ferulic acid, but no betanin, isobetanin and ascorbic acid as determined by HPLC. However, the peel and pulp contained high amounts of betanin, isobetanin and ascorbic acid, but with lower contents of phenolics and flavonoids as compared to the seed. These findings suggest that phenolics and flavonoids may directly contribute to the antioxidant activity of the seeds of ODHF.

Rice Albumin N-Terminal (Asp-His-His-Gln) Prevents against Copper Ion-Catalyzed Oxidations

Chromatographic separation of soluble proteins from rice (Oryza sativa L.) yielded a major albumin protein (16 kDa), with the DHHQVYSPGEQ sequence in the N terminus, showing antioxidant action. The rice albumin was more potent than other rice proteins in preventing Cu2+-induced low-density lipoprotein (LDL) oxidation. Additionally, it also exhibited a remarkable suppression of HOCl oxidation. In a further study, albumin inhibited Cu2+-induced oxidation of LDL in a stoichiometric manner with an EC50 value of 4.3 microM, close to that of serum albumins. Moreover, after digestion with trypsin or chymotrypsin, it maintained its antioxidant action. In an experiment to see the involvement of the N terminus in antioxidant action, a synthetic tetrapeptide, equivalent to the N terminus DHHQ, was found to inhibit Cu2+-induced LDL oxidation or degradation of apolipoprotein B, similar to that of rice albumin. In mechanistic analyses, the action of rice albumin or tetrapeptide is primarily due to the removal of Cu2+, as suggested from its inhibitory effect on Cu2+/diphenylcarbohydrazide (DPCH) complex formation. However, despite its similar inhibitory effect on Cu2+-induced oxidation of LDL, rice albumin was less effective than serum albumin in inhibiting Cu2+/DPCH complex formation, suggesting that the number of Cu2+-binding sites in rice albumin may be less than that in serum albumins. Taken together, rice albumin exerts a potent preventive action against Cu2+-induced oxidations, which is due to the Cu2+ binding by DHHQ in the N-terminal sequence. Such a role as a Cu2+ chelator would add up to the application of rice albumin protein.

Effect of loquat ( Eriobotrya japonica) extracts on LDL oxidation

The antioxidative activity of the extracts of loquat fruits, Eriobotrya japonica Lindley was examined. Loquat fruit was separated into three parts (peel, flesh and seed), and each part was extracted with either water or ethanol. The extracts were then assessed for their free radical scavenging ability and effects on the oxidation of human low density lipoprotein (LDL) in vitro. Results in this study show that the ethanol extracts of all three loquat parts and the water extract of the peels exhibited a strong ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. Among extracts of different loquat parts, the ethanol extract of loquat seeds was the most potent one. The ethanol extract of the seed was also effective in suppressing the oxidation of linoleic acid which was demonstrated by a slow discoloration of β-carotene/linoleic acid conjugation system. The ethanol extract of loquat seeds as compared to other extracts could also suppress significantly the 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN)-induced LDL oxidation. When the content of total polyphenolic compounds in different loquat parts (peel, flesh and seed) was examined, a significantly higher level of total polyphenols was found in the seed than the peels and flesh. Using reverse-phase HPLC-ESI EM analysis, significant levels of polyphenolic compounds such as chlorogenic acid, cyanidine glucoside, epicatechin, epigalocatechin gallate and procyanidin B 2 in the ethanol extract of different loquat parts were identified and quantified. The latter two compounds were found mainly in the ethanol extract of loquat seeds, but not in peels and flesh. Therefore, it is suggested that the high ability to scavenge free radicals and suppress the LDL oxidation exerted by the ethanol extract of loquat seeds was at least in part due to the high content of polyphenolic compounds in the seeds.

Tiliroside and gnaphaliin inhibit human low density lipoprotein oxidation

Two flavonoids, gnaphaliin and tiliroside, isolated from Helichrysum italicum, were studied in vitro for their capacity to inhibit Cu 2+-induced human low density lipoprotein (LDL) and diluted plasma oxidation. LDL oxidation was monitored by conjugated diene, thiobarbituric acid-reactive substances (TBARS) formation and electrophoretic mobility on agarose gel. Gnaphaliin and tiliroside increased the lag-phase for diene conjugate production in a dose-dependent manner. The reduction of TBARS production confirmed the antioxidant activity of gnaphaliin and tiliroside with 50% inhibitory concentration (IC 50) values of 8.0 ± 3.9 μM and 7.0 ± 2.6 μM respectively. Furthermore, the flavonoids negated the Cu 2+-induced increase in electrophoretic mobility of LDL. Antioxidant activity of gnaphaliin and tiliroside was significantly different when diluted plasma was oxidised by adding 1 mM CuSO 4. Although both flavonoids again reduced the TBARS production, tiliroside showed higher activity than gnaphaliin (IC 50 = 10.6 ± 2.5 μM vs. IC 50 > 50 μM). In conclusion, tiliroside and gnaphaliin are antioxidants against in vitro Cu 2+-induced LDL oxidation in the same order of magnitude compared to that of the reference drug, probucol.